Independent evolutionary origin of histone H3.3-like variants of animals and Tetrahymena.

All three genes encoding histone H3 proteins were cloned and sequenced from Tetrahymena thermophila. Two of these genes encode a major H3 protein identical to that of T. pyriformis and 87% identical to the major H3 of vertebrates. The third gene encodes hv2, a quantitatively minor replication independent (replacement) variant. The sequence of hv2 is only 85% identical to the animal replacement variant H3.3 and is the most divergent H3 replacement variant described. Phylogenetic analysis of 73 H3 protein sequences suggests that hv2, H3.3, and the plant replacement variant H3.III evolved independently, and that H3.3 is not the ancestral H3 gene, as was previously suggested (Wells, D., Bains, W., and Kedes, L. 1986, J. Mol. Evol., 23: 224-241). These results suggest it is the replication independence and not the particular protein sequence that is important in the function of H3 replacement variants.     

Regulation of the expression of histone H3.3 by differential polyadenylation.

Previously we have shown that the 3' untranslated regions (UTRs) of the replacement histone genes H3.3.A and H3.3B of Drosophila melanogaster differ in their nucleotide sequences and have different polyadenylation sites. To understand their functional relevance, which might explain the presence and evolutionary conservation of 2 different H3.3 genes, green fluorescent protein (GFP) constructs with different 3' UTR sections were studied by the expression of GFP as a marker protein. Here we show that the polyadenylation signals modify the cell-specific translation of the histone replacement variants in testes and ovaries. The H3.3A gene may be required to provide postmeiotic histone H3.3 in the male germ line in transition to chromatin packaging in sperm.     

Structure of a duck H3 variant histone gene: a H3 subtype with four cysteine residues

A duck recombinant DNA phage library was screened for H3 histone genes, and the sequence of a variant H3 gene, which appears not to be part of a histone gene cluster, has been determined. As derived from the nucleotide sequence, this gene codes for a 135-amino acid (aa) protein (as any other H3) and shows 10 aa substitutions compared with most published H3 structures. Six of these aa changes are based on one nucleotide (nt) substitutions in arginine codons. This results in three new histidines and, in addition to the highly conserved cysteine at position 110, three more cysteines are found in this H3 histone subtype.     

The localization of histone H3.3 in germ line chromatin of Drosophila males as established with a histone H3.3-specific antiserum

A rabbit antiserum, specific for the histone H3.3 replacement variant, was raised with the aid of a histone H3.3-specific peptide. Immuno blot experiments demonstrated the specificity of this polyclonal antiserum. In addition, we showed on immuno blots that two monoclonal antibodies isolated from mice with systemic lupus erythematosus (SLE) display strong reactivity with the H3.3 histone, but not with its replication-dependent counterparts. Our observations indicate that histone H3.3 might play a role as autoantigen in SLE. We used the histone H3.3-specific antiserum to characterize the germ line chromatin in cytological preparations of Drosophila testes, because our previous studies had shown that a histone H3.3-encoding gene is strongly expressed in the germ line of Drosophila males. The antiserum reacted with some of the lampbrush loops in spermatocytes and with chromatin of the postmeiotic germ cells of males. Our data indicate that histone H3.3 is not evenly distributed throughout the chromatin of germ cells, but is concentrated in distinct regions. Histone H3.3 disappears from the spermatid nuclei, along with the other core histones, during the late stages of spermatogenesis. In Drosophila polytene chromosomes, however, a rather uniform distribution of the histone H3.3 was observed. The possible role of histone H3.3 is discussed. Received: 12 May 1997 / Accepted: 4 July 1997     

DAXX co-folds with H3.3/H4 using high local stability conferred by the H3.3 variant recognition residues

Histone chaperones are a diverse class of proteins that facilitate chromatin assembly. Their ability to stabilize highly abundant histone proteins in the cellular environment prevents non-specific interactions and promotes nucleosome formation, but the various mechanisms for doing so are not well understood. We now focus on the dynamic features of the DAXX histone chaperone that have been elusive from previous structural studies. Using hydrogen/deuterium exchange coupled to mass spectrometry (H/DX-MS), we elucidate the concerted binding-folding of DAXX with histone variants H3.3/H4 and H3.2/H4 and find that high local stability at the variant-specific recognition residues rationalizes its known selectivity for H3.3. We show that the DAXX histone binding domain is largely disordered in solution and that formation of the H3.3/H4/DAXX complex induces folding and dramatic global stabilization of both histone and chaperone. Thus, DAXX uses a novel strategy as a molecular chaperone that paradoxically couples its own folding to substrate recognition and binding. Further, we propose a model for the chromatin assembly reaction it mediates, including a stepwise folding pathway that helps explain the fidelity of DAXX in associating with the H3.3 variant, despite an extensive and nearly identical binding surface on its counterparts, H3.1 and H3.2.     

Sequence and expression of the Drosophila phenylalanine hydroxylase mRNA

We report the cloning, nucleotide (nt) sequence and expression of the cDNA (pah) encoding phenylalanine hydroxylase (PAH) of Drosophila melanogaster. The strong hybridization signals observed in genomic blots when D. melanogaster DNA was probed with 32P-labeled human pah cDNA, indicated the existence of a high degree of sequence similarity between the pah genes of both species. The length of the pah genomic fragment is about 30 to 40 kb. The cDNA contains 84 bp of the 5'-untranslated region, 1359 bp of the protein-coding region and 87 bp of the 3' region, with only one polyadenylation signal. The isolated cDNA is probably full-length, since the size of the D. melanogaster PAH mRNA is 1.5 kb. At the nt level, the similarity of the D. melanogaster cDNA with human and rat pah cDNAs is 57.9% and 58.1%, respectively. The highest similarities are restricted to the nt sequence coding for the presumed hydroxylation domain. There is no nt sequence similarity between the first three exons of the human pah gene and an equivalent fraction of the D. melanogaster pah gene. At the amino acid (aa) level, the similarity in the presumed hydroxylation domain is 88.5%, in which two motifs of the structure AGLLSSXXXL are found, where X represents any aa. It was interesting to notice the conservation of aa 408, 311 and 280, where mutations are associated with phenylketonuria in humans. We observed, moreover, that, as it occurs in humans and rats, the expression of the D. melanogaster pah gene is tissue-specific and temporally regulated.     

组蛋白H3变体H3.3及其在细胞重编程中的作用

组蛋白是真核生物中一类进化上相对保守的蛋白质。由组蛋白八聚体及缠绕其上的DNA构成的核小体是真核生物染色质的基本组成单位。核小体使DNA保持固缩状态,既能维持基因组的稳定性,又能保证DNA序列可以正确地进行复制、转录、重组和修复。核小体调控细胞的生物过程除了通过组蛋白翻译后修饰,还可以通过组蛋白变体替换的方式进行。研究发现,组蛋白H3变体H3.3与常规组蛋白H3尽管仅有几个氨基酸的区别,但H3.3却能由特异的分子伴侣介导,整合进入染色质的特定区域,从而发挥不同的作用。同时,H3.3作为一种母源因子在正常受精和体细胞核移植等细胞重编程过程中也发挥着重要作用。本文总结了H3.3的结构特点和富集情况,探讨了特异的分子伴侣及其在细胞重编程中的作用,以期为提高体细胞重编程效率提供新思路,为体细胞重编程的应用奠定基础。     

Genes encoding a histone H3.3-like variant in Arabidopsis contain intervening sequences.

Two genes encoding a particular H3 histone variant were isolated from Arabidopsis thaliana. These genes differ from the H3 genes previously cloned from Arabidopsis and other plants by several interesting properties: (1) the two genes are located close to each other; (2) their coding regions are interrupted by two or three small introns, the two closest to the initiation codon being located at the same place in the two genes; (3) another, long intron is located in the 5'-untranslated region just before the initiation codon of gene I as deduced from the sequence of several corresponding cDNAs, and very likely also of gene II; (4) these genes do not show preferential expression in organs containing meristematic tissues contrary to the classical intronless replication-dependent histone genes, thus suggesting that their expression is not replication-dependent; (5) the protein encoded by both genes is the same and corresponds to a minor H3 variant highly conserved among all the plant species studied up to now. All these characteristics are common with the animal replication-independent H3.3 histone genes and it is assumed that the genes described here are the first example of the equivalent H3.3 gene family in plants. Interestingly, the promoter regions of the two genes have the same general structure as the Arabidopsis intronless genes. Possible implications on the regulation of H3 genes expression are discussed.     

组蛋白变体H3.3 L27M突变与胶质瘤发生

组蛋白变体在基因表达等基本细胞过程中发挥重要调节功能。人类有5种H3变体,分别为H3.1、 H3.2、H3.3、着丝粒特异性CENP-A和睾丸特异性H3t。人H3.3有H3F3A和H3F3B两个基因编码。采用DNA全基因组测序的方法在儿童高级别胶质瘤如恶性胶质瘤（GBM）和弥漫性内在脑桥胶质瘤（DIPG）鉴定出高频的H3F3A突变。超过70%DIPG和30%GBM携带H3.3 K27M氨基酸错义突变（27位赖氨酸被甲硫氨酸代替）。H3.3 K27M通过与组蛋白H3K27甲基转移酶EZH2亚基相互作用而抑制多梳抑制复合物2（PRC2）活性并全面减少H3K27me3含量。因此H3.3 K27M突变重塑了表观修饰状态和基因表达模式,从而驱动肿瘤发生。K27M突变可作为分子标志物以更好区分儿童胶质瘤亚型,还可作为特异、敏感的预后标志物。通过抑制组蛋白去甲基化酶如JMJD3活性而增加H3K27甲基化可作为K27M突变胶质瘤治疗的有效策略。本文综述了组蛋白变体H3.3 K27M在胶质瘤中的突变模式、分子机制和临床应用。     

Expression of a mouse replacement histone H3.3 gene with a highly conserved 3'' noncoding region during SV40- and polyoma-induced Go to S-phase transition.

We have isolated and sequenced a mouse replacement variant histone H3.3 cDNA. It corresponds to the most abundant mRNA expressed from a unique gene by the use of one out of three polyadenylation sites. The 3' non coding region of H3.3 is very long (approximately 1100 nt) and highly conserved throughout evolution since it is about 95% homologous to the 3' non coding region of the chicken H3.3B gene. We studied the expression of the H3.3 gene during SV40- and polyoma-induced mitotic host reaction in confluent, Go-arrested primary mouse kidney cell cultures. H3.3 replacement variant mRNA steady state levels increased during the Go to S-phase transition, apparently as the result of two mechanisms: one related to cell growth, whereas the other was linked to cellular DNA synthesis. The latter mechanism was however far less pronounced than with replication histone variant mRNAs. The biological implications of these results are discussed.     

Molecular turnover,the H3.3 dilemma and organismal aging (hypothesis)

The H3.3 histone variant has been a subject of increasing interest in the field of chromatin studies due to its two distinguishing features. First, its incorporation into chromatin is replication independent unlike the replication‐coupled deposition of its canonical counterparts H3.1/2. Second, H3.3 has been consistently associated with an active state of chromatin. In accordance, this histone variant should be expected to be causally involved in the regulation of gene expression, or more generally, its incorporation should have downstream consequences for the structure and function of chromatin. This, however, leads to an apparent paradox: In cells that slowly replicate in the organism, H3.3 will accumulate with time, opening the way to aberrant effects on heterochromatin. Here, we review the indications that H3.3 is expected both to be incorporated in the heterochromatin of slowly replicating cells and to retain its functional downstream effects. Implications for organismal aging are discussed.     

Human histone acetyltransferase 1 protein preferentially acetylates H4 histone molecules in H3.1-H4 over H3.3-H4

In mammalian cells, canonical histone H3 (H3.1) and H3 variant (H3.3) differ by five amino acids and are assembled, along with histone H4, into nucleosomes via distinct nucleosome assembly pathways. H3.1-H4 molecules are assembled by histone chaperone CAF-1 in a replication-coupled process, whereas H3.3-H4 are assembled via HIRA in a replication-independent pathway. Newly synthesized histone H4 is acetylated at lysine 5 and 12 (H4K5,12) by histone acetyltransferase 1 (HAT1). However, it remains unclear whether HAT1 and H4K5,12ac differentially regulate these two nucleosome assembly processes. Here, we show that HAT1 binds and acetylates H4 in H3.1-H4 molecules preferentially over H4 in H3.3-H4. Depletion of Hat1, the catalytic subunit of HAT1 complex, results in reduced H3.1 occupancy at H3.1-enriched genes and reduced association of Importin 4 with H3.1, but not H3.3. Finally, depletion of Hat1 or CAF-1p150 leads to changes in expression of a H3.1-enriched gene. These results indicate that HAT1 differentially impacts nucleosome assembly of H3.1-H4 and H3.3-H4.     

Constitutive expression, not a particular primary sequence, is the important feature of the H3 replacement variant hv2 in Tetrahymena thermophila.

Although quantitatively minor replication-independent (replacement) histone variants have been found in a wide variety of organisms, their functions remain unknown. Like the H3.3 replacement variants of vertebrates, hv2, an H3 variant in the ciliated protozoan Tetrahymena thermophila, is synthesized and deposited in nuclei of nongrowing cells. Although hv2 is clearly an H3.3-like replacement variant by its expression, sequence analysis indicates that it evolved independently of the H3.3 variants of multicellular eukaryotes. This suggested that it is the constitutive synthesis, not the particular protein sequence, of these variants that is important in the function of H3 replacement variants. Here, we demonstrate that the gene (HHT3) encoding hv2 or either gene (HHT1 or HHT2) encoding the abundant major H3 can be completely knocked out in Tetrahymena. Surprisingly, when cells lacking hv2 are starved, a major histone H3 mRNA transcribed by the HHT2 gene, which is synthesized little, if at all, in wild-type nongrowing cells, is easily detectable. Both HHT2 and HHT3 knockout strains show no obvious defect during vegetative growth. In addition, a mutant with the double knockout of HHT1 and HHT3 is viable while the HHT2 HHT3 double-knockout mutant is not. These results argue strongly that cells require a constitutively expressed H3 gene but that the particular sequence being expressed is not critical.